Red cell transfusions are associated with inflammation and thrombosis, both arterial and venous, the mechanisms of which are not understood. Platelets are critically involved in hemostasis, thrombosis and inflammation. Our hypothesis is that storage-induced changes in red cells including the release of free hemoglobin and the accumulation of biologically active mediators in the supernatant have a direct deleterious effect on the recipient's platelets. We propose that unwanted effects on platelets contribute to the inflammatory and pro-thrombotic properties of red cell transfusions. We will study changes in stored leukoreduced Adsol (AS)-1 red cells and their supernatants over time, and ascertain the effects of supernatant mediators and stored red cells on recipient platelets. In Aim 1, red cell-free hemoglobin and key lipid mediators that accumulate in microparticles and supernatant fractions of leukoreduced red cells will be defined, and we will ascertain the effects of stored red cells and these mediators on platelets. We will determine whether FDA licensed manipulations, such as washing and/or rejuvenation, ameliorate the effects of stored red cells and supernatant on platelet function. Mediators that will be studied based on our preliminary and published data include: hemoglobin which alters platelet function; F2 isoprostanes that are markers of oxidative stress and cell damage and directly affect platelet function; and red cell-derived microparticles which stimulate platelets. Assessment of biologic effects on platelets will employ aggregation, spreading and bioactive/proinflammatory mediator release. We will focus our investigations on red cell supernatant mediators because our supporting studies demonstrate that supernatant removal reduces morbidity and mortality in patients with hematologic malignancies. Our compelling preliminary laboratory data demonstrate that even leukoreduced red cells accumulate large amounts of mediators (hemoglobin, isoprostanes and microparticles) in the supernatant that alter platelet function in vitro and if transfused may alter recipient platelet function and promote vascular disease and inflammation. In Aim 2, we will investigate the clinical importance of red cell storage time and infused mediators by correlating these with clinical and laboratory outcomes in critically ill patients. A prospective cohort study of 600 ICU patients will characterize the relationship of infused cell-free hemoglobin, isoprostanes, prostaglandins, and red cell storage time, with laboratory and clinical outcome measures of inflammation and hemostasis/thrombosis. Laboratory measures include CD40 ligand, C-reactive protein, D-dimer, blood cell count and differential. Clinical outcomes include organ failure, bleeding, thrombosis, time on ventilator and length of hospital stay. Overall, the proposed studies will determine whether the adverse effects of red cell transfusions are likely due to the stored red cells, to accumulated supernatant mediators, or to both. PUBLIC HEALTH RELEVANCE: Red cell transfusions are one of the most commonly used therapies in hospitalized patients. Transfusions are associated with increased complications, including lung failure, blood clots and heart attack. Our research will discover explanations for these effects, and we will devise inexpensive strategies to reduce these complications